SPECTROSCOPIC AND ELECTROCHEMICAL STUDIES ON ACTIVE-SITE TRANSITIONS OF THE TYPE-1 COPPER PROTEIN PSEUDOAZURIN FROM ACHROMOBACTER CYCLOCLASTES

The single type 1 copper protein pseudoazurin from Achromobacter cyclo clastes gives reversible electrochemical behavior at a (4-pyridyl) dis ulfide-modified gold electrode. Measurements carried out at 25.0 degre es C indicate a midpoint reduction potential of E(1/2) = 260 mV versus normal hydrogen electrode at pH 7.0 and a peak-to-peak separation of Delta E(p) = 59 mV. The diffusion coefficient and heterogeneous electr on transfer rate constant are estimated to be 2.23 x 10(-6) cm(2) s(-1 ) and 3.7 x 10(-2) cm s(-1), respectively. Also, controlled potential electrolysis indicates a 1-electron transfer process and a formal redu ction potential of 259 mV versus normal hydrogen electrode for the Cu( II)/Cu(I) couple. The heterogeneous electron transfer rate constant de termined at the (4-pyridyl)disulfide-modified gold electrode at pH 4.6 is 6.7 x 10(-3) cm s(-1), consistent with a slower process at the pos itively charged electrode surface. At pH 11.3, UV-visible, EPR, and re sonance Raman spectra indicate a conversion of the distorted tetrahedr al copper geometry to a trigonal structure. The trigonal form has elon gated axial bonding and an axial EPR spectrum. At pH 11.3, the reducti on potential is further decreased, and Cu-S bands in resonance Raman s pectra at 330-460 cm(-1) are shifted to higher energy (similar to 10 c m(-1)), consistent with a stronger Cu-S bond.